1. Field of the Invention
This invention relates to reagents useful in the preparation of 5'-biotinylated oligonucleotides.
2. Summary of the Background
Oligonucleotides and single-stranded DNA bearing reporter groups are generally referred to as nucleic acid probes. Nucleic acid probes can be used to detect nucleotide sequences of interest in DNA or RNA by specific hybridization and have proved extremely valuable for a variety of uses, including gene localization and the detection of mutations. Such probes are therefore useful as diagnostic tools for both research and clinical purposes. The detection of nucleic acid sequences by nucleic acid probes can be carried out using standard methods, such as "Southern Hybridization," "Sandwich Hybridization," "In situ Hybridization," or the "Dot Blot" technique.
Although high specific activity .sup.32 P has commonly been used as the reporter group for nucleic acid probes, the use of this radioisotope is problematic from both a logistical and a health standpoint. The short half-life of .sup.32 P necessitates the anticipation of reagent requirements several days in advance and prompt use of such a reagent. Once .sup.32 P-tagged nucleic acid probes have been generated, they are prone to self-destruction and must be immediately used in hybridization assays. Subsequent autoradiography required for visualization of the labeled probes is a slow process (overnight exposures are common). Finally, possible health risks are associated with the use and disposal of such potent radioisotopes. To address these problems, replacement of .sup.32 P/ autoradiography with alternative, nonradioisotopic reporter/detection systems has been considered.
The most common nonradioisotopic reporter group used in nucleic acid probes is (+)-biotin (vitamin H). This is due in particular to its safety and the sensitivity of the systems developed for its detection. (+)-Biotin forms very tight (K.sub.D =10.sup.-15 M) complexes with the proteins avidin and streptavidin, which can be easily visualized by enzyme-based systems, many of which are commercially available. All such systems are based on avidin or streptavidin chemically bound to an enzyme that catalyzes a reaction generating an easily detectable substance. Preferred enzymes include alkaline phosphatase, beta galactosidase, horseradish peroxidase, and luciferase. These enzyme systems produce substances that are easily detected by their color, fluorescence, or luminescence. (+)-Biotin is [3aS-(3a.alpha.,4.beta., 6a.alpha.)]-hexahydro-2-oxo-1H-thieno[3,4-d]-imidazole-4-pentanoic acid; however, it is the hexahydro-2-oxo1H-thieno[3,4-d]-imidazole portion of the molecule that is responsible for its binding to avidin and streptavidin. Hereafter, "biotinylated" substances and substances containing the "biotin group" refer to substances containing hexahydro-2-oxo-1H-thieno[3,4-d]-imidazole.
Biotinylated nucleic acid probes can either be long (generally &gt;100 nucleotides) or short (generally 8-30 nucleotides) in length, and both types have particular uses and advantages. Long biotinylated nucleic acid probes have been prepared by the multiple incorporation of biotinylated nucleoside triphosphates by nick translation using a DNA polymerase or by 3'-tailing with multiple biotinylated nucleosides using terminal deoxynucleotide transferase, see Ward, et al., U.S. Pat. No. 4,711,955. Drawbacks to these enzymatic methods for preparing biotinylated probes include the expense of biotinylated nucleoside triphosphates and the need for obtaining the probe's sequence from a natural source.
Short nucleic acid probes ("oligonucleotide probes") have two major advantages over long probes. First, they are much more sensitive to small numbers of base mismatches with their complementary target DNA, making them particularly useful, for example, in detecting mutations. Second, although short probes cannot be easily synthesized enzymatically, they can be conveniently prepared in large quantities (10-100 nanomoles) by introducing a reporter group onto readily available synthetic oligonucleotides. For example, short radioactive probes are most typically prepared by enzymatically labeling synthetic oligonucleotides with .sup.32 P.
The preferred method of synthesizing oligonucleotides is by solid phase synthesis using either the phosphotriester, phosphoramidite, or hydrogen phosphonate approach. Most oligonucleotides are now prepared very conveniently by commercially available automated DNA synthesizers, all of which use the phosphoramidite approach or, more recently, the H-phosphonate approach.
A number of methods have been described for the preparation of synthetic biotinylated oligonucleotide probes. In one general approach, an oligonucleotide possessing an added group with unusual reactivity, for example, an aliphatic amino group, is first prepared either by solid phase synthesis or by a combination of solid phase and solution techniques. The purified oligonucleotide possessing the unusually reactive group is treated with a reactive derivative of (+)-biotin to afford the biotinylated probe, which is then further purified. Examples of this and related approaches have been disclosed by Chollet, et al., Nucleic Acids Res. 13, 1529-41 (1985); Wachter, et al., Nucleic Acids Res. 14, 7985-94 (1986); Agrawal, et al., Nucleic Acids Res. 14, 6227-45 (1986); Urdea, et al., Nucleic Acids Res. 16, 4937-56 (1988); and Cook, et al., Nucleic Acids Res. 16, 4077-95 (1980). All of these approaches to biotinylated oligonucleotide probes require specialized reagents, as well as considerably more time, effort, and chemical expertise than does automated chemical synthesis.
There are two examples of methods for preparing biotinylated oligonucleotide probes in which all reactions, including the final biotin attachment step, are performed on a solid support. Carr, et al., European Patent Application No. 86302750.4 (publ. 1986) have disclosed a method for preparing 5'-biotinylated oligonucleotides by reacting the deblocked 5'-hydroxy group of a solid supported oligonucleotide with a phosphorylated derivative of (+)-biotin in the presence of a condensing reagent. Kempe, et al., Nucleic Acids Res. 13, 45-57 (1985) have described a similar approach in which the deblocked 5'-hydroxy group of a solid supported oligonucleotide is first treated with p-chlorophenyl-phosphoditriazolide, the remaining phosphorous-triazole bond is hydrolyzed, and finally the phosphorylated oligonucleotide is treated with 2-(biotinylamido)ethanol in the presence of a condensing reagent. Although these two approaches have the general advantages of solid phase synthesis, they require reagents, solvents, and reaction conditions that are not commonly used in commercial automated DNA synthesizers. Consequently, solid supported oligonucleotide probes prepared using these methods on an automated DNA synthesizer must be biotinylated manually.
The purpose of the present invention is to overcome the disadvantages encountered in the prior art by providing biotinylating reagents useful for directly preparing 5'-biotinylated oligonucleotides. The reagents of the present invention make 5'-biotinylated oligonucleotides as accessible as ordinary oligonucleotides and are compatible with automated DNA synthesizers that utilize phosphoramidite chemistry.